Glucose controls utilization and metabolic fates of carbohydrates in prokaryotes and eukaryotes. Knowledge of molecular mechanisms of this control is important for understanding basic aspects of biology and many pathological states in humans. Protein-protein interactions are the basis for most biological regulation, including much of glucose signaling. This work focuses on protein-protein interactions in molecular mechanisms of allosteric control by which signals that are generated by glucose uptake and metabolism regulate carbon source metabolism and fate in bacteria. The studies focus on allosteric control of members of the sugar kinase/actin/hspTO superfamily of enzymes. A central issue is determining whether the motions of the layered structure of the conserved ATPase catalytic core of superfamily members, which are believed to be part of the normal catalytic cycle, are the basis for allosteric control. The issue is addressed by comparing the allosteric control of E. coli glycerol kinase by IIAGIc, a phosphotransferase system protein, to that of superfamily members that have gained IIAGIc control as a result of molecular evolution in the laboratory. Evolved enzymes that show a remarkable range of IIAGIc binding affinity and inhibition efficacy have been prepared. Additional enzymes will be evolved from the absolutely non-allosteric superfamily member, E. coli xylulokinase, by using combinatorial mutagenesis and molecular breeding methods. A powerful positive genetic selection will be used to identify the target enzymes from libraries of candidates. The energetic of thermodynamic coupling will be determined from steady state kinetics and ligand binding studies by using the approach of linked functions. The static and dynamic structures will be determined by X-ray crystallography and fluorescence methods. IIAGIc allosteric control of glycerol kinases displays behavior that is not seen for classical allosteric enzymes, activation with respect to substrate binding but inhibition with respect to enzyme catalysis. The energetics of such non-classical allosteric control has not been described. These studies will reveal the role of the conserved catalytic core structure and the active site closure conformational change in allosteric control of superfamily members and the molecular basis for glucose control of carbohydrate metabolism in E. coli.